Immunoglobulin (Ig) heavy chain variable (V) region diversity is generated by several mechanisms including the multiplicity and combinatorial assortment of germline V region gene segments (VH, D, and JH), junctional diversification mechanisms at the VH-D and D-JH junctions (CDR3), and somatic mutation. To date, studies of the aged human antibody repertoire have not yet focused on the molecular mechanisms which generate Ig heavy Ig heavy chain diversity. In this proposal it is postulated that alterations in these mechanisms may contribute to the decline in immunocompetence and increase in autoantibodies associated with aging in humans. Human VH region diversity in the aged will be examined by construction of VH region CDNA libraries from B cells using an approach which randomly amplifies all VHCmu RNA transcripts independent of VH, D, and J segment composition. VH family-specific probes will then be used to quantify the representation of each of the six human VH gene families. The usage of specific VH members within families will be ascertained by hybridization analysis using gene- specific oligonucleotides and by DNA sequencing. Potential biases in D and JH segment usage or in the association of particular V,D, and J segments in aged VH-regions will be examined by DNA sequence analysis. The contribution of N-regions and P nucleotides to the CDR3 regions of expressed H-chain rearrangements will be assessed. Potential inefficiency in Ig gene recombination in the aged could be masked by examination of only expressed H-chain rearrangements since productive alleles have presumably undergone selection. Therefore, we will also analyze genomic VH-region libraries generated from DNA using primers (VH and JH) which will amplify both productive and no-productive rearrangements. Finally, the extent of somatic mutation in VH5H-chain CDNAS amplified from splenic germinal center cells will be evaluated in young and aged adults. Definitive identification of somatic mutations will be facilitated by the simplicity (1-2 functional members) and lack of polymorphism in the VH5 family. Knowledge of the molecular basis of VH region diversity is critical to an understanding of generation of the human antibody repertoire in both young and aged individuals.